A high-performance liquid chromatographic method with on-line post-column photochemical derivatization and fluorimetric detection for the simultaneous separation and quantitative determination of aflatoxin (AF) B1 , B2 , G1 , and G2 in foodstuffs and feed materials is reported. The chromatographic separation is accomplished by using a C18 column eluted with an isocratic mobile phase consisting of water, methanol, and acetonitrile. The sample preparation requires a simple extraction of aflatoxins with a mixture of water and methanol, and a purification step by immunoaffinity column clean-up. The total analysis time, including sample preparation and chromatographic separation, does not exceed 40 min with a run time of 10 min. The procedure for the determination of aflatoxins in food samples and cereals for animal consumption has been extensively validated, in agreement with Regulation (EC) No. 882/2004, demonstrating the conformity of the method with provisions of Regulation (EC) No. 401/2006 in terms of sensitivity, linearity, selectivity, and precision.